Rapamycin (sometimes called sirolimus) was first described in 1975 as an antifungal agent isolated from Streptomyces hygroscopicus (Vezina, 1975; Sehgal, 1975). In 1987, the structurally related compound FK506 (sometimes called tacrolimus) was characterized as a potent immunosuppressive agent (Tanaka, 1987), and shortly thereafter, rapamycin was also shown to have potent immunosuppressive activity. In spite of rapamycin's immunosuppressive activity and structural similarity to FK506, the two compounds suppress the immune response in completely different ways (Schreiber, 1992). FK506 inhibits the T cell receptor (TCR) signal and prevents activation of a resting helper T cell. Rapamycin inhibits the autocrine signaling pathway involving interleukin-2 (IL-2) and the IL-2 receptor (IL-2R). These latter signals commit the cell to a program of cell division by communicating with the components of the cell cycle machinery necessary for DNA replication.
Both FK506 and rapamycin are potentially useful in the treatment of human disease. FK506 has been approved by the FDA for use in treating the rejection of transplanted organs. A similar use has been envisioned for rapamycin, and its demonstrated activity in organ transplantation and autoimmune animal models indicate a high clinical potential. Rapamycin has been shown to have antitumor activity against B16 melanocarcinoma, colon 26 tumor, EM ependymoblastoma, CD8F1 mammary and colon 38 murine tumors (Sehgal, 1993). Rapamycin has also shown immunosuppressive activity in assays to measure prevention of development of autoimmune adjuvant arthritis, experimental allergic encephalomyelitis and autoimmune uveoretinitis in the rat (Sehgal, 1993).
The biological activity and structural novelty of both rapamycin and FK506 led to a search for their cellular target(s), and the target of both compounds was identified as the plentiful cytoplasmic protein FKBP12 (for FK506 binding protein) of 12 kDa molecular mass. Since FK506 and rapamycin bound to the same target (Kd of 0.4 and 0.2 nM, respectively) and affected different pathways, a new function was attributed to the FKBP12-ligand complex. This new function arises from the ability of FKBP12-FK506 and FKBP12-rapamycin complexes, but not the individual components, to bind to and inhibit still other protein targets. The FKBP12-FK506 complex inhibits the phosphatase activity of calcineurin, a crucial component of the TCR pathway. Calcineurin is a serine/threonine phosphatase also called PP2B. The FKBP12-rapamycin complex inhibits the IL-2R signal by binding to a large (289 kDa) protein named FRAP in humans (Brown et al, 1994) or RAFT in rats (Sabatini et al, 1994; Chiu et al, 1994).
The structural basis for the tight binding of FK506 and rapamycin by FKBP12 has been investigated by both X-ray diffraction and NMR techniques (Clardy, 1995). In particular, high resolution X-ray structures are available for FKBP12-FK506 (1.4 .ANG. resolution) and FKBP12-rapamycin (1.7 .ANG. resolution) (Van Duyne et al, 1991; Van Duyne et al, 1991a; Van Duyne et al, 1993). These structures reveal, among other things, the fold of FKBP12, the atomic details of the hydrophobic binding pocket, and the details of how FK506 and rapamycin interact with the binding pocket. A structural analysis of the complex formed between FKBP12-FK506-caldneurin is also available (Griffith et al, 1995). That structure reveals how the portion of FK506 not involved in binding FKBP12 interacts with calcineurin and inhibits its phosphatase activity.
The biochemical characterization of FRAP, the target of the FKBP12-rapamycin complex, remains incomplete. The C-terminal domain resembles a phosphatidylinositol (PI) kinase, but to date no PI or protein kinase activity has been convincingly demonstrated. FRAP (RAFT, TOR) are members of a rapidly growing and important family of proteins that have been identified only recently (Zakian, 1995). ATM, TEL1, DNA-PK and MEC1 are some of the recently characterized members of this family of PIK-related kinases. (See e.g., Keith, 1995). ATM (for ataxia telngiectasia mutant) is responsible for a human autosomal hereditary disease characterized by cerebellar degeneration, progressive mental retardation, uneven gait, dilation of blood vessels, immune deficiencies, premature aging and a hundredfold increase in cancer susceptibility (Zakian, 1995). Persons who are heterozygous in ATM are believed to be at elevated risk for cancer. Mutations to TEL1 lead to abnormally short telomeres, and in conjunction with other mutations can lead to sensitivity to X-rays, UV radiation and hydroxyurea. DNA-PK is, as the name suggests, a DNA-dependent protein kinase that recognizes damaged DNA, and human cells without DNA-PK activity are radiation sensitive and repair deficient. MEC1 is required for both S-M and G2-M checkpoint progression as well as for meiotic recombination in yeast. Thus MEC1 is arguably the master checkpoint gene in yeast.
FRAP is a large protein (2549 amino acid residues), and only a small fraction can be involved in recognizing the FKBP12-rapamycin complex. Fortunately all of these residues are in one domain, and this domain, which is called the FKBP12-rapamycin binding (FRB) domain, is the protein used in this invention. It was identified through tryptic digests of FRAP and independently produced as an 11 kDa soluble protein (Chen et al, 1995)
Unfortunately, until now, three-dimensional structural details of the association of FKBP12-rapamycin with the FRB domain of FRAP have remained completely unknown. In the absence of such three-dimensional structural details, it has been impossible to design compounds based on that structure which would be capable of mimicking rapamycin's binding to the FRB domain. We have now obtained crystals of that ternary complex and have determined its three dimensional structure. With this information, it is now possible for the first time to rationally design compounds capable of binding to an FRB domain and mimicking the pharmacological activity of rapamycin. Such mimics may be used in place of rapamycin as immunosuppressive agents or in other pharmacological applications.